Assays for detection of viral envelope-mediated fusion of an enveloped virion with its host cell has received significant attention since this step represents one of the earliest events in the viral life cycle. Assays for detection of infection of host cells by human immunodeficiency virus (HIV), the etiologic agent of acquired immunodeficiency syndrome (AIDS), is of particular interest. HIV, a member of the lentivirus family of retroviruses, primarily infects helper T lymphocytes and monocytes/macrophages, i.e. cells that express surface CD4, leading to a depletion of CD4+ T lymphocytes.
The initial phase of the HIV replicative cycle involves the high affinity interaction between the HIV exterior envelope glycoprotein gp120 and the cellular receptor CD4 (Klatzmann, D. R., et al., Immunodef., Rev. 2, 43–66 (1990)). CD4 is sufficient to render otherwise infection-resistant cells susceptible to HIV infection (Maddon, P. J., et al., Cell 47, 333–348 (1986)). cDNA clones encoding HIV gp120 and CD4 have been isolated (Maddon, P. J., et al., Cell 42, 93–104 (1985), Wain-Hobson, S., et al., Cell 40, 9–17 (1985)).
Following the binding of HIV-1 gp120 to cell surface CD4, viral and host cell, the HIV gp41, the transmembrane component of the envelope glycoprotein, facilitates fusion of the viral and target cell membranes, resulting in introduction of the viral capsid into the target cell cytoplasm (Maddon, P. J., et al., Cell 54:865 (1988); Kowalski, M., et al., Science 237:1351 (1987)).
Methods for detecting viral fusion events mediated by a viral envelope proteins, such as the HIV-1 envelope glycoprotein, are useful in, for example, identification of agents which alter viral envelope protein-mediated cell fusion.
Conventional assays to examine viral fusion events have principally depended on the use of cell-to-cell fusion assays in which receptor-expressing target cells are cultured with other cells engineered to express the relevant viral envelop gene product. For example, fusion of HIV-1 virions with the host cell plasma membrane has been mimicked by assaying fusion of HIV-1 infected cells expressing gp120/gp41, or by assaying fusion of cells stably expressing recombinant gp120/gp41 with uninfected CD4+ cells (see, e.g., Lifson, J. D., et al., Nature 323:725 (1986)). Although such cell-to-cell fusion studies have yielded valuable information, they do not fully recapitulate all of the variables governing the fusion of actual virions to cellular targets. For example, the density and distribution of the HIV envelope proteins may differ significantly between natural virions and cells engineered to express the HIV-1 env gene products. Furthermore, fusion of HIV in certain cell types such as macrophages (Marchal, V., et al., J. Virology 75:11166–11177 (2001)), or by certain strains of HIV-1 such as SF2 (Fackler, O. T. et al., Curr. Biol. 10:1005–1008 (2000)), may occasionally occur after initial viral entry by macropinocytosis or endocytosis respectively. Thus the assay does not actually detect membrane fusion, but rather only the presence of the virion in the cell within an intracellular compartment, e.g., the viral envelope has not actually fused to provide for delivery of the viral genome to the host cell cytoplasm. Therefore, such processes cannot be recapitulated in cell-to-cell fusion assays.
Only a virion-based fusion assay, applicable to study of viral fusion in primary cells can account for all of these variables that influence the fusion process. Lowy et al. and Chen et al. describe a virion-based fusion assay based on the redistribution of a self-quenching flurophore (Lowy, R. J., et al., PNAS 87:1850–1854 (1990); Chen, Y. D., et al., Biophys. Chem. 34:283–292 (1989)), Raviv et al. describes a virion-based fusion assay based on photosensitized activation of a hydrophobic probe by a fluorescent lipid loaded into the target membrane (Raviv, Y., et al., Virology 293:243–251 (2002)). These assays are complex, and thus far have not been adapted to study fusion in complex cell populations. Muthumani et al. describes a virion-based fusion assay based on detection of fluorescence from a Vpr-GFP fusion protein present in the virion (Muthumani et al. DNA Cell Biol. 19:179–188 (2000). However this assay does not distinguish between virion entry by endocytosis (a non-productive route of infection) and actual fusion (the productive route of viral entry). Distinguishing between the productive fusion route of entry and the non-productive endocytic route is important, particularly since, in some cells, up to 90% of virions entering the cells do so by the non-productive endocytic route. Thus so far no simple and rapid virion-based fusion assay has been developed.
There still exists a need for a virion-based fusion assay that provides for high sensitivity and specificity for detection of viral fusion events, including detection of a relatively low number of viral fusion events. There is also a need for a virion-based fusion assay that can be applied to detection of viral fusion in biologically relevant cellular targets present in complex cell populations, and which provides for detection using a variety detection methods, such as flow cytometry, fluorescence microscopy, and UV photometry. In addition, the assay should be rapid, reproducible, and quantitative, adaptable to various enveloped virus types and various cell types, and relatively safe (e.g., does not require high titers of enveloped virus).
The present invention addresses these needs.